Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to α4ß7.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

Mechanisms of HIV protein degradation into epitopes: implications for vaccine design.

The degradation of HIV-derived proteins into epitopes displayed by MHC-I or MHC-II are the first events leading to the priming of HIV-specific immune responses and to the recognition of infected cells. Despite a wealth of information about peptidases involved in protein degradation, our knowledge of epitope presentation during HIV infection remains limited. Here we review current data on HIV protein degradation linking epitope production and immunodominance, viral evolution and impaired epitope presentation. We propose that an in-depth understanding of HIV antigen processing and presentation in relevant primary cells could be exploited to identify signatures leading to efficient or inefficient epitope presentation in HIV proteomes, and to improve the design of immunogens eliciting immune responses efficiently recognizing all infected cells.

Drug use and the risk of HIV infection amongst injection drug users participating in an HIV vaccine trial in Bangkok, 1999-2003.

BACKGROUND: HIV spread rapidly amongst injecting drug users (IDUs) in Bangkok in the late 1980s. In recent years, changes in the drugs injected by IDUs have been observed. We examined data from an HIV vaccine trial conducted amongst IDUs in Bangkok during 1999-2003 to describe drug injection practices, drugs injected, and determine if drug use choices altered the risk of incident HIV infection. METHODS: The AIDSVAX B/E HIV vaccine trial was a randomized, double-blind, placebo-controlled trial. At enrolment and every 6 months thereafter, HIV status and risk behaviour were assessed. A proportional hazards model was used to evaluate demographic characteristics, incarceration, drug injection practices, sexual activity, and drugs injected during follow-up as independent predictors of HIV infection. RESULTS: The proportion of participants injecting drugs, sharing needles, and injecting daily declined from baseline to month 36. Amongst participants who injected, the proportion injecting heroin declined (98.6-91.9%), whilst the proportions injecting methamphetamine (16.2-19.6%) and midazolam (9.9-31.9%) increased. HIV incidence was highest amongst participants injecting methamphetamine, 7.1 (95% CI, 5.4-9.2) per 100 person years. Injecting heroin and injecting methamphetamine were independently associated with incident HIV infection. CONCLUSIONS: Amongst AIDSVAX B/E vaccine trial participants who injected drugs during follow-up, the proportion injecting heroin declined whilst the proportion injecting methamphetamine, midazolam, or combinations of these drugs increased. Controlling for heroin use and other risk factors, participants injecting methamphetamine were more likely to become HIV-infected than participants not injecting methamphetamine. Additional HIV prevention tools are urgently needed including tools that address methamphetamine use.

Engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes.

Broadly neutralizing anti-HIV antibodies are rare and have proved hard to elicit with any immunogen. We have tested in vitro the notion that such antibodies or other antiviral proteins could be made by lentivirus-mediated gene transfer into human hematopoietic stem/progenitor cells (HSPCs), followed by differentiation of the transduced cells into B cells, the most potent antibody-producing cells. To do this, we have developed a highly efficient system for in vitro maturation of secreting B lymphocytes and plasma cells from CD34(+) HSPCs. It is a 3-stage, in vitro culture system that supports normal human B-lineage development from HSPCs to antibody-secreting plasmablasts (approximately 36%) and plasma cells (approximately 20%). By transducing human cord blood CD34(+) cells with lentiviral vectors encoding a secretory monoclonal anti-HIV antibody, b12 (IgG(1)), we were able to program human B cells to produce in vitro up to 1.5 microg/mL of this broadly neutralizing antibody. Our results suggest that an HIV vaccine might be delivered by autologous transplantation of in vitro-programmed HSPCs, which would develop into antibody-secreting B cells in vivo and provide a continuous supply of anti-HIV neutralizing antibodies.

DNA gag/adenovirus type 5 (Ad5) gag and Ad5 gag/Ad5 gag vaccines induce distinct T-cell response profiles.

Results from Merck's phase II adenovirus type 5 (Ad5) gag/pol/nef test-of-concept trial showed that the vaccine lacked efficacy against human immunodeficiency virus (HIV) infection in a high-risk population. Among the many questions to be explored following this outcome are whether (i) the Ad5 vaccine induced the quality of T-cell responses necessary for efficacy and (ii) the lack of efficacy in the Ad5 vaccine can be generalized to other vector approaches intended to induce HIV type 1 (HIV-1)-specific T-cell responses. Here we present a comprehensive evaluation of the T-cell response profiles from cohorts of clinical trial subjects who received the HIV CAM-1 gag insert delivered by either a regimen with DNA priming followed by Ad5 boosting (n = 50) or a homologous Ad5/Ad5 prime-boost regimen (n = 70). The samples were tested using a statistically qualified nine-color intracellular cytokine staining assay measuring interleukin-2 (IL-2), tumor necrosis factor alpha, macrophage inflammatory protein 1beta, and gamma interferon production and expression of CD107a. Both vaccine regimens induced CD4(+) and CD8(+) HIV gag-specific T-cell responses which variably expressed several intracellular markers. Several trends were observed in which the frequencies of HIV-1-specific CD4(+) T cells and IL-2 production from antigen-specific CD8(+) T cells in the DNA/Ad5 cohort were more pronounced than in the Ad5/Ad5 cohort. Implications of these results for future vaccine development will be discussed.

Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference.

HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

Advances in methods for the production, purification, and characterization of HIV-1 Gag-Env pseudovirion vaccines.

HIV pseudovirion or virus-like particle vaccines represent a promising approach for eliciting humoral and cellular immune responses. Pseudovirions present the envelope glycoprotein complex in its authentic trimeric form, and thus have the potential to generate neutralizing antibodies against relevant virion-associated epitopes that may be lacking in protein subunit vaccines. The development of pseudovirion particles as a viable vaccine approach for progression to clinical testing has been limited by a number of factors, including shedding of particle-associated gp120, practical limitations to large-scale production and purification, and the generation of antibodies against cellular proteins incorporated on the particle surface that confound the analysis of HIV-specific neutralizing antibody responses. Here, we review methods that address each of these challenges, with a focus on production methods for generating non-infectious Gag-Env pseudovirions. Mammalian cell lines that inducibly express HIV Gag and Env can overcome production limitations, and produce pseudovirions that retain gp120 following purification. Baculovirus production systems have the potential to provide higher quantities of particles, but cleavage of gp160 remains a current limitation. Anti-cellular antibody responses can be diminished by adsorption with cell lysates or whole cells. These technical advances should facilitate the further development of pseudovirion vaccine approaches in preclinical testing and future clinical trials.

Optimization of the delivery of heterologous proteins by the Salmonella enterica serovar Typhimurium type III secretion system for vaccine development.

Type III protein secretion systems, which are organelles with the capacity to deliver bacterial proteins into host cells, have been adapted to deliver heterologous antigens for vaccine development. A limitation of these antigen delivery systems is that some proteins are not amenable to secretion through this pathway. We show here that proteins from the simian and human immunodeficiency viruses that are not permissive for secretion through a Salmonella enterica serovar Typhimurium type III secretion system can be modified to travel this secretion pathway by introduction of discrete mutations. Proteins optimized for secretion were presented more efficiently via the major histocompatibility complex class I pathway and were able to induce a better immune response.

Oral delivery of replication-competent adenovirus vectors is well tolerated by SIV- and SHIV-infected rhesus macaques.

Although replication-competent adenovirus (Ad) vectors are promising in AIDS vaccine design, their safety in immune compromised hosts is unknown. To initially address this question, enteric-coated tablets containing a replicating Ad vector were orally administered to SHIV- and SIV-infected rhesus macaques with normal, intermediate or low CD4 T cell counts and stable disease. The vector was detected within a week after tablet administration in stools of all animals but not in nasal secretions, indicating no spread of virus to the upper respiratory tract. CD4 T cell counts and viral loads remained stable in all animals and no signs of fever, weight loss, or other clinical symptoms of Ad-induced disease were observed during 10 weeks of follow-up. Oral delivery of the replicating Ad vector was safe and well tolerated by SHIV- and SIV-infected hosts. Oral enteric-coated tablets may prove safe for administering replicating Ad-vectored vaccines in areas with high HIV prevalence.

Biochemical and immunogenic characterization of soluble human immunodeficiency virus type 1 envelope glycoprotein trimers expressed by semliki forest virus.

The current lack of envelope glycoprotein immunogens that elicit broadly neutralizing antibody responses remains a major challenge for human immunodeficiency virus type 1 (HIV-1) vaccine development. However, the recent design and construction of stable soluble gp140 trimers have shown that some neutralization breadth can be achieved by using immunogens that better mimic the functional viral spike complex. The use of genetic delivery systems to drive the in vivo expression of such immunogens for the stimulation of neutralizing antibodies against HIV-1 may offer advantages by maintaining the quaternary structure of the trimeric envelope glycoproteins. Here, we describe the biochemical and immunogenic properties of soluble HIV-1 envelope glycoprotein trimers expressed by recombinant Semliki Forest virus (rSFV). The results presented here demonstrate that rSFV supports the expression of stable soluble gp140 trimers that retain recognition by conformationally sensitive antibodies. Further, we show that rSFV particle immunizations efficiently primed immune responses as measured after a single boost with purified trimeric gp140 protein, resulting in a Th1-biased antibody response. This differed from the Th2-biased antibody response obtained after repeated immunizations with purified gp140 protein trimers. Despite this difference, both regimens stimulated neutralizing antibody responses of similar potency. This suggests that rSFV may be a useful component of a viral vector prime-protein boost regimen aimed at stimulating both cell-mediated immune responses and neutralizing antibodies against HIV-1.

Evidence as a HIV-1 self-defense vaccine of cyclic chimeric dodecapeptide warped from undecapeptidyl arch of extracellular loop 2 in both CCR5 and CXCR4.

Novel conformation-specific antibodies were raised against a cyclic chimeric dodecapeptidyl multiple antigen peptide (cCD-MAP) constructed with a spacer-armed Gly-Asp dipeptide and two pentapeptides (S(169)-Q(170)-K(171)-E(172)-G(173) of CCR5 and E(179)-A(180)-D(181)-D(182)-R(183) of CXCR4) which are components of the undecapeptidyl arch (UPA: from R(168) to C(178) in CCR5, from N(176) to C(186) in CXCR4) of extracellular loop 2 (ECL2) in chemokine receptors (CCR5 and CXCR4). Of the antibodies raised, one monoclonal antibody, CPMAb-I (IgMkappa), reacted with cCD-MAP, but not with the linear chimeric dodecapeptide-MAP. The antibody reacted with the cells separately expressing CCR5 or CXCR4, but not with those not expressing the coreceptors. Moreover, the antibody markedly suppressed infection by X4, R5, or R5X4 virus in a dose-dependent manner in a new phenotypic assay for drug susceptibility of HIV-1 using CCR5-expressing Hela/CD4(+) cell clone 1-10 (MAGIC-5). Moreover, CPMAb-I interfered with LAV-1(BRU) infection (m.o.i. = 0.01) of Molt4#8 cells cocultured with CPMAb-I-producing hybridoma in the transwell, and significantly interfered with neither chemotaxis nor calcium influx induced with stromal cell-derived factor 1 alpha (SDF-1alpha). Thus, the antibody raised against the cCD-MAP provides powerful protection or defense against HIV-1 infection. We therefore propose the cCD-MAP or its derivative immunogen as a novel candidate for an HIV-1 coreceptor-based self-defense vaccine.

Pronounced acute immunosuppression in vivo mediated by HIV Tat challenge.

HIV infection is accompanied by an early immune dysfunction limiting host control of virus and likely contributing to difficulties in achieving a successful vaccine against HIV. We report here that the HIV Tat protein is strongly immunosuppressive, both immediately after immunization of mice with soluble protein (sTat), and in seroconverting humans, and propose that Tat-induced suppression cripples immune surveillance to HIV infection. We show that macrophages are sensitive to sTat stimulation at concentrations 1,000-fold lower (500 pM) than T cells, and this stimulation is accompanied by the immunosuppressive induction of Fas ligand on the macrophage. T cell proliferative defects induced by sTat in vitro can be completely (at lower concentrations of sTat) or partially (at higher concentrations) reversed by antagonists to Fas/Fas ligand interaction. We further report a method to preserve immunogenicity while inactivating Tat immunosuppression through oxidation, which advances the use of oxidized Tat as a component of an anti-HIV vaccine. These observations define additional methods to study the immunosuppressive functions of sTat that now may be rapidly applied to primary isolates from individuals with differing clinical courses. Our findings have immediate relevance for vaccine development, by describing and supporting a strategy that includes inactivated sTat in a multicomponent, anti-HIV vaccine.

Immunization of mice with peptomers covalently coupled to aluminum oxide nanoparticles.

Subunit vaccines generally require adjuvants to elicit immune responses, but adjuvants may alter the conformation of critical epitopes and reduce vaccine efficacy. We therefore tested an immunization strategy in which antigen is covalently coupled to aluminum oxide nanoparticles using a method that favors preservation of the native conformation. The test antigen consisted of "peptomers" (head-to-tail-linked peptide homopolymers) derived from the 4th conserved region (C4) of HIV-1 gp120 which is believed to be in an alpha-helical conformation prior to binding to CD4. Immune responses in mice to peptomer-nanoparticle conjugates were compared to responses elicited by free C4 peptide and C4 peptomers, with and without the hydrophilic adjuvant muramyl dipeptide (MDP). Highest peptomer-specific serum antibody responses were induced by peptomer-particles without MDP. Serum antibodies induced by peptomer-particles also showed highest reactivity towards recombinant, glycosylated gp120 and HIV-1 infected T cells. The results suggest that this novel vaccine approach could be useful for induction of immune responses against conformation-sensitive viral antigens without the need for additional adjuvants.

Increase in the immunogenicity of HIV peptide antigens by chemical linkage to polytuftsin (TKPR40).

The use of synthetic peptide antigens in human prophylaxis still suffers from the very important problem of finding suitable carriers devoid of side effects. A desirable carrier for use in humans would be poorly immunogenic by itself, yet it would enhance the immune response to the peptide antigen. In the study reported herein, we examined the role of polytuftsin (TKPR40), a synthetic polymer of the natural immunomodulator tuftsin, as a carrier for synthetic peptides of HIV derived from the gp41 and gp120 proteins. Chimeric immunogens were constructed by chemical linkage between synthetic peptides of HIV and polytuftsin. These were employed for immunization of mice of different MHC haplotypes, and the humoral and cellular immune responses developed against the peptides were assessed by measuring total IgG, IgG, subclasses, T-cell proliferation, and in vitro cytokine release. A significantly stronger immune response was observed in mice immunized with the peptide-polytuftsin conjugates than in mice receiving the peptide dimers (peptide-peptide). Peptide-polytuftsin conjugates induced IgG2a and IgG2b isotype switching after both primary and secondary immunization. In addition, there was a positive correlation between the amounts of cytokines and the shift in the IgG isotypes. These data suggest that the use of polytuftsin as a carrier may increase the immune response against poorly immunogenic synthetic peptides.